Heparosan polymers and methods of making and using same for the enhancement of therpeutics

ABSTRACT

Compositions, methods, and systems for the development and use of heparosan as a therapeutic modifying agent or vehicle which can modulate drug cargo pharmacokinetics and behavior within a mammalian patient.

CROSS-REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

The present application is a continuation of U.S. Ser. No. 15/597,824,filed May 17, 2017, which is a divisional of U.S. Ser. No. 12/556,324,filed Sep. 9, 2009; which claims benefit under 35 USC § 119(e) ofprovisional application U.S. Ser. No. 61/179,275, filed May 18, 2009.The '324 application is also a continuation-in-part of U.S. Ser. No.12/383,046, filed Mar. 19, 2009; which claims benefit under 35 USC §119(e) of provisional applications U.S. Ser. No. 61/095,572, filed Sep.9, 2008; and U.S. Ser. No. 61/038,027, filed Mar. 19, 2008. The entirecontents of each of the above-referenced patents and patent applicationsare hereby expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract NumberMCB9876193 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND 1. Field of the Invention

The presently disclosed and/or claimed inventive concept(s) disclosedand/or claimed inventive concept(s) relates, in general, to the field oftherapeutics and, more particularly but without limiting, to novelcompositions and methods for making heparosan biomaterials that aresuitable for conjugation to therapeutics for the purpose of enhancingdrug action and/or delivery as well as bioreactive agents forbiotechnical applications.

2. Brief Description of the Related Art

Without limiting the scope of the presently disclosed and/or claimedinventive concept(s), the background of the related art is described inconnection with the use of sugar polymers and, more particularly,heparosan as a therapeutic modifying and/or coupling agent.

The presently disclosed and/or claimed inventive concept(s) relatesgenerally to the field of therapeutics and, more particularly, to thedevelopment of enhanced therapeutics through the use of modifying and/orcoupling agents and, in particular but without limitation, naturalpolysaccharides and oligosaccharides such as heparosan. A wide range ofexisting and near-term therapeutics has great potential, but manypossess drawbacks that slow or prevent implementation for aiding humanhealth. Fortunately, the physical, chemical, and/or biological nature ofa promising drug candidate may sometimes be assisted by modifying theparental drug. A widely used agent, poly[ethylene glycol] (PEG) has beenapproved by the Food & Drug Administration (FDA) for use withtherapeutic “cargo” including small molecule drugs, polypeptides, andliposomes, for example. The process of adding PEG to a drug, i.e.,“PEGylation,” has been very successful, as shown in Table 1. Thehydrophilic chains of PEG polymers increase the solubility of the cargoin water, protect the cargo when in the human body and prolong thetherapeutic action of the cargo. Due to its artificial nature, itschemical synthesis, and its potential harmful effects when ingested inlarge quantities over long periods of time, the use of PEG hassignificant drawbacks and alternatives have been sought. The presentlydisclosed and claimed invention is directed to such alternativemodifying and/or coupling agents, which overcome the defects anddisadvantages of the prior art.

DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 graphically depicts the structures of heparosan and polyethyleneglycol.

FIG. 2A is a graphical representation of the pharmacokinetics (pK) ofradioactive heparosan conjugate in plasma in a rat model. Rats wereinjected intravenously with ¹²⁵I-heparosan polymer (100 kDa mass) at‘Time 0’, and at various times, blood was drawn, and the radioactivityin the plasma was measured. The data indicate that 100 kDa heparosan,the active molecule of HEPylation, has a long lifetime (half-life ofapproximately 2 days) in the mammalian bloodstream.

FIG. 2B is a graphical representation of the pharmacokinetics ofradioactive heparosan conjugate in plasma in a rat model. Rats wereinjected intravenously with ¹²⁵I-heparosan polymer (60 kDa monodispersepolymer) at ‘Time 0’, and at various times, blood was drawn, and theradioactivity in the plasma was measured. The data indicate that 60 kDaheparosan, the active molecule of HEPylation, has a long lifetime(half-life of approximately 15 hours) in the mammalian bloodstream. Inaddition, upon comparison of the data in FIGS. 2A and 2B, the size ofthe polymer determines the plasma half-life, thus allowing tuning ofdrug-conjugate pharmacokinetics.

FIG. 3 is a graphical representation of the fate of a radioactiveheparosan conjugate in a rat model. Rats were injected intravenouslywith 100 kDa ¹²⁵I-heparosan polymer at ‘Time 0’, and at various times,the radioactivity in blood (Plasma or red and white blood cells, ‘R & WBC’), organs (liver, kidney, spleen, heart, bladder, brain), andexcreted waste (urine, feces) was measured. The data indicate thatheparosan, the active molecule of HEPylation, circulated in the plasmaof the mammalian blood stream, did not accumulate in major organs (note:the low signal present is due to blood trapped in organs based onsaline-perfused controls), and was excreted via normal pathways (i.e.,urine, feces).

FIG. 4 is a pictorial representation showing heparosan is very stable inthe mammalian bloodstream. The 100 kDa ¹²⁵I-heparosan conjugate wasinjected intravenously into rats, and at various times, blood waswithdrawn, and the plasma was isolated. The samples were deproteinizedand analyzed by agarose gel (1.5%) electrophoresis and autoradiography.The molecular weight of starting probe (lane H; arrow) and the polymerin plasma samples are equivalent even after approximately 1-2 days time.Over time, the polymer is removed from circulation within the mammal andthen metabolized/excreted.

FIG. 5 is graphical representation showing synthesis of monodisperseheparosan polymers. Three batches of heparosan polymer were analyzed ona 1.2% agarose gel with Stains-all detection. The polymer size isreadily controlled (as indicated by the three different size bands of800 kDa, 380 kDa, and 100 kDa from top to bottom). The tight bandsindicate that the products have a narrow size distribution(polydispersity M_(w)/M_(n)=1.06 to 1.18; for reference, the value of anideal monodisperse polymer is 1). The size of the polymer affects itshalf-life in the bloodstream; thus, HEPylation is a means of tuningtherapeutic dosing profiles. In addition, the Food & Drug Administration(FDA) regulatory hurdles for production and approval of therapeutics arelower for a more defined, monodisperse molecule in comparison to a lessdefined, polydisperse molecule.

FIG. 6 is a graphical representation of one strategy for HEPylationreagent preparation and utilization to form a therapeutic conjugate.Three or four sequential reactions are used to produce a HEPylated cargoin this embodiment, where activated heparosan vehicle is coupled to acargo. I. An acceptor (a heparosan tetrasaccharide, Hep₄) is modified toadd a reactive group (e.g., R=amino or hydrazide). II. Three independentreaction mixtures, each with a different ratio of UDP-sugar/reactiveacceptor, are elongated with PmHS1 synthase via polymer grafting toyield a set of distinct reactive monodisperse heparosan polymers ofthree different sizes. III. The Cargo is modified directly with any onesize polymer. III'. Alternatively (dotted line), an additionalmodification step is used to alter the reactivity of the heparosanreagent (e.g., add a R′=maleimide group onto amino-heparosan) allowingthe next step, IV, modification of Cargo. Alternative embodimentsinclude (a) activating heparosan produced by fermentation of bacteriaand then coupling to cargo or (b) coupling the activated short acceptorto a cargo, then elongating via polymer grafting to a useful, desiredsize heparosan chain with heparosan synthase PmHS1.

FIG. 7 are pictorial representations of SDS-PAGE gels illustrating theproduction of HEPylated BSA molecules (left panel) and degradationthereof with heparosan lyase (right panel).

FIG. 8 are pictorial representations of an SDS-PAGE gel (left panel) andgel filtration chromatography profile (right panel) illustratingproduction of a series of higher molecular weight products correspondingto a series of HEPylated BSA molecules.

FIG. 9 is a pictorial representation of an SDS-PAGE gel illustrating theproduction of HEPylated IgG molecules (see arrow area).

FIG. 10 is a pictorial representation of a PAGE gel visualized by virtueof ultraviolet-induced fluorescence, demonstrating production ofHEPylated fluorescein molecules (see arrow). Unreacted FITC (fluoresceinisothiocyanate) is bracketed.

FIG. 11 is a graphical representation of thin layer chromatography (TLC)of short heparosan acceptor coupled to a radioactive cargo and itssubsequent elongated product. This TLC shows the new radioactiveacceptor formed by coupling BH and amino-HEP4 (middle lane) and itssubsequent elongation by polymer grafting with PmHS1 synthase into aheparosan vehicle (left lane) suitable for prolonging residence time inthe mammalian blood stream. (BH=¹²⁵I Bolton-Hunter reagent;Hep4=heparosan tetrasaccharide; Poly=BH conjugate of heparosan polymerof approximately 220 kDa).

FIG. 12 is a graphic depiction (right panel) of the production ofHEPylated cargo by polymer grafting, and a pictorial representation(left panel) of an SDS-PAGE gel that demonstrates the use of said methodto produce HEPylated BSA molecules (see bracket area).

FIG. 13 is a pictorial representation of an SDS-PAGE gel demonstratingthe production of HEPylated BSA molecules (see bracket area) utilizingnaturally occurring heparosan obtained from in vivo microbialfermentation as the source of the vehicle.

FIG. 14 is a graphical representation of an agarose gel analysis ofheparosan coupled to radioactive cargo. This gel was stained with asugar detection reagent (Stains-all) as well as exposed to X-ray film(Autorad; 2 exposure times—short or long) to illustrate the definedsynthesis of a radioactive cargo coupled to approximately 220 kDaheparosan (same polymer as in the TLC of FIG. 11). The narrow sizedistribution (monodispersity) is demonstrated by loading of both a low(1×) and a high (10×) concentration of HEPylated probe as well asoverexposure of the X-ray film.

FIG. 15 is a graphical representation of the total distribution of themeasured radioactivity from FIG. 16 for i.m. dosing.

FIG. 16 is a graphical representation illustrating the curve-fit for pKanalysis of the elimination of a radioactive heparosan compound from theplasma for i.m. dosing.

FIG. 17 is a pictorial representation of an agarose gel demonstratingthe stability of the heparosan vehicle utilized in FIGS. 15-16 in theextracellular compartments of the mammalian body.

FIG. 18 is a graphical representation of TLC of Urine Metabolites. Aradioactive conjugate of approximately 220 kDa heparosan/Bolton-Hunterreagent was injected into a rat. After 2 days, the radioactive breakdownproducts excreted into urine were analyzed by TLC (similar to FIG. 11).Small size fragments (equal or less than 4 sugar units or n=2) of theoriginal probe are observed, indicating metabolic breakdown of thenatural heparosan polymer after leaving the bloodstream (note: thisapproximately 220 kDa conjugate before injection into the rat wouldremain at the origin of the TLC plate as in FIG. 11 and not run up theTLC plate as shown here).

FIG. 19 is a graphical representation of heparosan metabolite excretioninto feces. A conjugate of approximately 220 kDa,heparosan/Bolton-Hunter was injected into a rat. Over 2 days time, theradioactive breakdown products excreted into feces were measured in agamma counter and plotted here as the percent fraction of the entireinitial dose. Excretion into feces and urine accounts for themetabolized heparosan vehicle indicating that heparosan does notaccumulate in a mammalian patient (as shown in FIG. 3). The size of theradioactive polymers in the feces was less than 3 kDa (or less than 15sugar units) as measured by ultrafiltration, thereby indicating thatheparosan is degraded and then excreted over time.

DETAILED DESCRIPTION

Before explaining in detail at least one embodiment of the invention indetail by way of exemplary drawings, figures, graphs, tables, andexperimental drafts, for example, it is to be understood that thepresently disclosed and/or claimed inventive concept(s) is not limitedin its application to the details of construction and the arrangement ofthe components set forth in the following description or illustrated inthe drawings. The presently disclosed and/or claimed inventiveconcept(s) is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting. While the making andusing of various embodiments of the presently disclosed and/or claimedinventive concept(s) are discussed in detail below, it should beappreciated that the presently disclosed and/or claimed inventiveconcept(s) provide many applicable inventive concepts that can beembodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the presently disclosed and/or claimed inventive concept(s)and do not delimit the scope of the invention.

The needs of the presently disclosed and/or claimed inventive concept(s)set forth above as well as further and other needs and advantages of thepresent invention are achieved by the embodiments of the inventiondescribed herein below.

The presently disclosed and/or claimed inventive concept(s) provides forthe improvement and enhancement of therapeutics through the conjugationand use of a novel therapeutic modifying agent: heparosan, a naturalpolysaccharide related to heparin. Heparosan can be synthesized in astep-wise, reproducible, and defined manner so as to provide all of theadvantages of PEG without its potential side effects. Heparosan issoluble in water, biocompatible, and bio-inert within the human body.

The addition of heparosan (HEP) to a therapeutic cargo molecule, aprocess termed herein as “HEPylation”, is superior to PEGylationbecause: a) a larger size range of heparosan polymers is more readilysynthesized than PEG; b) the size distribution at longer chain lengthsof heparosan can be controlled more carefully than PEG; c) heparosan hasa higher water solubility than PEG; d) as a naturally occurringpolysaccharide, heparosan's degradation products are biocompatible; ande) heparosan is not immunogenic.

Several linear and branched PEGs having different molecular weights havebeen employed by those with skill in the art to improve thepharmacokinetic behavior of therapeutic drugs (i.e., the “cargo” carriedby the PEG molecule). Several distinct types of reactive PEG polymersallow the synthesis of both reversible and irreversible PEG-drugconjugates. PEG-drug conjugates typically exhibit prolonged residence invivo, decreased degradation by metabolic enzymes, and reducedimmunogenicity. The therapeutic cargo, including proteins and peptides,small molecule drugs, and liposomes, have been PEGylated and evaluatedsuccessfully by the FDA (Table 1). Several PEGylated drugs have been inuse for more than a decade, thus proving the general applicability andsafety of PEGylation. As shown and claimed herein, therapeutic cargothat has been HEPylated (i.e., conjugated to heparosan) retain all ofthe benefits of PEGylated cargo while minimizing the negative andundesirable attributes of PEG.

TABLE 1 Currently Marketed PEGylated Drugs (adapted from‘Pharmacotherapy (2003) 23 (8 pt 2): 3S-8S) Generic Name (TradeBioactivity Cargo of PEG Name)/Manufacturer of Native Main Effect ofReason Conjugate (FDA Approval Date) Agent Pegylation for Treatment ADA(adenosine Pegademase (ADAGEN/ Enzyme Longer half-life, SCID (severedeaminase) Enzon (March 1990) replacement, reduced immune combinedreverses symptoms response immunodeficiency of ADA deficiency disease)Asparaginase Pegasparagase Hydrolyzes Longer half-life In combination(ONCASPAR/ asparagine, on reduced immune chemotherapy for Enzon(February 1994) which leukemic response treatment of acute cells aredependent lymphoblastic leukemia in patients hypersensitive to L-asparaginase Granulocyte Pegfilgrastim (NEULASTA/ Stimulation of Longerhalf-life, Prophylaxis against colony- Amgen (January 2002) neutrophilself-regulating severe neutropenia stimulating production clearance andits complications factor during myelosuppressive chemotherapy Interferonα2b Peginterferon α2b Antiviral cytokine Slower clearance, Hepatitis Cin patients (PEGASYS/Roche sustained serum with compensated (October2002) concentration liver disease Stealth PEG Pegylated liposomalAntitumor Slower clearance, Refractory ovarian liposomes withdoxorubicin (CAELYX anthracycline greater distribution cancer, Kaposi'sdoxorubicin DOXIL/Alza (June 1999) into tumors sarcoma

Various sized PEGs circulate and are cleared out of the bloodstream ofmammals at different periods of times. As shown in Table 2, PEG polymershaving a molecular weight of 6,000 Da or 6 kDa (PEG-6) have a shorterhalf-life in blood serum than PEG polymers having a molecular weight of170,000 Da or 170 kDa (PEG-170). The half-life of PEG molecules in bloodserum is directly dependent upon the size of the polymer. Even thoughPEGylation may lead to a loss in binding affinity due to stericinterference (due to the PEG chain partially covering the drug surfaceand by conjugating with some of the drugs active sites) with thedrug-target binding interaction, the loss in potency is offset by thelonger half-life of the PEG drug conjugate circulating in the bloodstream. Certain drugs have, therefore, been enabled for use byconjugating the drug to PEG and thereby increasing its half-life withina patient to be treated that otherwise could not have been developed.Much effort is currently ongoing with the goal of improving orre-tooling existing drugs by conjugating with PEG. The novel use ofheparosan for a PEG replacement is, therefore, a significant stepforward and is providing a highly biocompatible and targeted drugdelivery device.

TABLE 2 Blood circulation of PEG (adapted from ‘J. Phar. Pharm. Sci.’2000 (3): 125-136) Parameter PEG-6 PEG-20 PEG-50 PEG-170 AUC 6.2 110 6001110 t_(1/2), minutes 17.6 170 990 1390 AUC = area under the curve;t_(1/2) = half life

The main pharmacokinetic outcomes of PEGylation are summarized aschanges occurring in the overall circulation life-span within bloodserum, tissue distribution pattern, and elimination pathway of the drugPEG conjugate (Table 3). As with PEG, heparosan maintains all of thebenefits of PEG while improving bio-compatibility and the ability toselectively produce and target polymers of a desired predetermined size(Sismey-Ragatz et at., J. Biol. Chem, 2007). As with PEG conjugation,conjugation of a drug or therapeutic molecule with heparosan (1)increases retention of the drug in the circulation by protecting againstenzymatic digestion, (2) slows filtration by the kidneys, and (3)reduces the generation of neutralizing antibodies. In all respects,HEPylation is a clear substitute for PEGylation and, as a naturallyoccurring polysaccharide, brings with it an enhanced biocompatibilityand simpler sugar conjugation chemistry.

TABLE 3 Beneficial Features of Therapeutic Modifying Agents: PEGylationversus HEPylation PEG Heparosan HEPylation a. Extend Cargo Half-iife inBloodstream? Yes Yes (e.g., avoid renal clearance if larger molecularweight) b. Protect Cargo from Degradation? Yes Yes (e.g., by proteases)c. Shield Cargo from Immune Response? Yes Yes (e.g., prevent antibodygeneration) d. Trap Cargo in Cancerous Regions? Yes Yes (e.g., due toaltered tumor vasculature) e. Enhance Solubility of Cargo? Yes Yes,increased solubility potential (e.g., especially hydrophobicchemotherapy agents)? than PEG due to its more hydrophilic nature f.Variety of Cargo Coupling Chemistries? Yes Yes (e.g., amine, sulfhydrylreactive) g. Exhance Cargo Stability? Yes Yes (e.g., prevent proteinunfolding events) h. Reduce Dosage and Maintain Constant Yes Yes BloodConcentrations? (e.g., avoid peaks and troughs; predictable dosingplateau in desired range) i. Suitability for a Range of Cargo: (e.g.,platform technology) proteins, peptides? Yes Yes small MW drugs? Yes Yesliposomes? Yes Yes hormones? Yes Yes

First, the nature of degradation of artificial PEG may be a limitingfactor for pharmaceuticals used at high doses and/or for long durationtreatments. Second, the quality control of PEG polymer synthesis withrespect to molecular weight distribution is not as great as desired.

Certain carbohydrates play roles in forming and maintaining thestructures of multicellular organisms in addition to more familiar rolesas nutrients for energy. Glycosaminoglycans (GAGS) are long linearpolysaccharides comprising disaccharide repeats that contain an aminosugar. GAGs are well known to be essential in vertebrates.

The GAG structures possess a significant number of negative groups andhydroxyl groups and are, therefore, highly hydrophilic. Depending on thetissue and cell type, the GAGs are structural, adhesion, and/orsignaling elements in humans. A few microbes also produce extracellularpolysaccharide coatings called capsules that are composed of GAG chainsand that serve as virulence factors. The capsule assists in themicrobe's evasion of host defenses such as phagocytosis and complement.As the microbial polysaccharide is identical or very similar to the hostGAG, the antibody response to the microbe is either very limited ornon-existent.

In humans, polymers of heparosan (also called N-acetylheparosan orunsulfated, unepimerized heparin;[4-GlcUA-beta-1,4-GlcNAc-alpha-1-]_(n); shown in FIG. 1) only existtransiently, serving as a precursor to the more highly modified finalproducts of heparan sulfate and heparin. The bacterial-derived enzymesused to produce heparosan for use in one embodiment of the presentlydisclosed and/or claimed inventive concept(s) synthesize heparosan astheir final product. A single polypeptide, the heparosan synthase PmHS1of Pasteurella multocida Type D, polymerizes the heparosan sugar chainby transferring both GlcUA and GlcNAc. PmHS1 is a robust enzyme thatefficiently makes polymers up to ˜1 MDa (1,000 kDa or ˜5,000monosaccharide units) in vitro. In Escherichia coli K5, at least twoenzymes, KfiA, the alpha-GlcNAc transferase, and KfiC, thebeta-GlcUA-transferase, (and perhaps KfiB, a protein of unknownfunction) work in concert to form the disaccharide repeat of heparosan.The E. coli enzyme complex is not as efficient as the PmHS1 enzyme as itis more difficult to produce the long polymer chains with the E. colienzyme complex. However, for the purpose of the presently disclosedand/or claimed inventive concept(s), it is intended and would beunderstood by one of skill in the art that any method which producesheparosan may be used. It is not the method of producing heparosan thatis determinative—rather, it is the conjugation of heparosan from anysource or method of production (e.g., fermented heparosan produced bynative or recombinant microbes, as well as chemoenzymatic syntheses ororganic chemical syntheses) to a target molecule (i.e., the cargo) forincreased solubility in water, bioavailability and dwell time within thepatient that is presently disclosed and claimed.

A key advantage to using heparosan is that it has increased biostabilityin the extracellular matrix when compared to other GAGs such ashyaluronic acid and chondroitin. As with most compounds synthesized inthe body, new molecules are typically made, and after serving theirpurpose, are broken down into smaller constituents for recycling.

Heparin and heparan sulfate, for example, are degraded by a singleenzyme known as heparanase. Experimental challenge of heparosan andN-sulfa-heparosan with heparanase, however, shows that since thesepolymers lack the O-sulfation of heparin and heparan sulfate, heparosanand N-sulfo-heparosan are not sensitive to enzymatic action in vitro byheparanase. These findings indicate that heparosan is not fragmentedenzymatically in the body, thereby indicating that heparosan is a stablebiomaterial for use as a drug conjugate.

However, if heparosan or any of its fragments (generated by reactiveoxygen species, etc.) is internalized into the lysosome, then themolecules will be degraded by resident beta-glucosidase andbeta-hexosaminidase enzymes (which remove one sugar at a time from thenon-reducing termini of the GAG chain), similar to the degradation ofheparin or hyaluronic acid. Therefore, the heparosan polymer isbiodegradable and will not permanently reside in the body and therebycause a lysosomal storage problem. A key advantage for therapeuticmodification with heparosan polymer, HEPylation, is that normalmonosaccharides, GlcNAc and GlcUA are the products of the eventualdegradation. In contrast, PEG degrades into reactive artificialaldehydes and ketones which are toxic above certain levels. PEG alsoaccumulates in the body, especially when present as one or more highmolecular weight polymers.

The normal roles of heparin/heparan sulfate in vertebrates includebinding coagulation factors (inhibiting blood clotting) and growthfactors (signaling cells to proliferate or differentiate). The keystructures of heparin/heparan sulfate that are recognized by thesefactors include a variety of O-sulfation patterns and the presence ofiduronic acid [IdoUA]; in general, polymers without these modificationsdo not stimulate clotting or cell growth. Heparosan-based materialswhich do not have such O-sulfation patterns, therefore, do not provokeunwanted clotting or cellular growth/modulation. As such, HEPylated drugconjugates do not initiate clotting and/or cell growth processes andremain solely bio-reactive as per the drug or cargo constituent-theheparosan is thus termed or deemed to be biologically inert.

Foreign or unnatural molecules stimulate the immune system. Heparosanpolymer exists transiently during heparan sulfate and heparinbiosynthesis as well as being found in very short polymer structureswithin mature heparan sulfate or heparin chains. In the latter case, theN- and O-sulfation reactions are not complete in mammals, so traces ofthe original heparosan remain; for example, approximately 1-5 unsulfateddisaccharide repeats can be interspersed within the sulfated regions.Therefore, the body treats heparosan as ‘self,’ and does not mount animmune response. P. multocida Type D and E. coil K5 utilize heparosancoatings to ward off host defenses by acting as molecular camouflage.Indeed, scientists had to resort to using capsule-specific phages orselective GAG-degrading enzymes to type these heparosan-coated microbessince a conventional antibody or serum could not be generated—theheparosan is thus termed or deemed non-immunogenic or non-antigenic.

To facilitate the understanding of the presently disclosed and/orclaimed inventive concept(s), a number of terms are defined below. Termsdefined herein have meanings as commonly understood by, a person ofordinary skill in the areas relevant to the presently disclosed and/orclaimed inventive concept(s). Terms such as “a”, “an” and “the” are notintended to refer to only a singular entity, but include the generalclass of which a specific example may be used for illustration. Theterminology herein is used to describe specific embodiments of theinvention, but their usage does not delimit the invention, except asoutlined in the claims. Generally, all technical terms or phrasesappearing herein (unless defined explicitly differently herein) are usedas one skilled in the art would understand to be their ordinary meaning.It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the presently disclosed and/or claimed inventiveconcept(s), and vice versa. Furthermore, compositions of the presentlydisclosed and/or claimed inventive concept(s) can be used to achievemethods of the presently disclosed and/or claimed inventive concept(s).

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich the presently disclosed and/or claimed inventive concept(s)pertains. All publications and patent applications (including issuedpatents) are herein incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this specification, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure.

Heparosan is a sugar polymer of the formula-[GlcNAc-alpha4-GlcUA-beta4]_(n)- where n is from 2 to about 5,000. Theterm “oligosaccharide” generally denotes n being from about 1 to about11 while the term “polysaccharide” denotes n being equal to or greaterthan 12. The term “conjugate” as used herein refers to a complex createdbetween two or more compounds by covalent or weak bonds. The term“cargo” as used herein refers to the drug, therapeutic or otherbiologically active component in the conjugate, while the term “vehicle”as used herein refers to the carrier of the cargo (e.g., the heparosanpolymer) in the conjugate.

As used herein, the term “active agent(s),” “active ingredient(s),”“pharmaceutical ingredient(s),” “therapeutic,” “medicant,” “medicine,”“biologically active compound” and “bioactive agent(s)” are defined asdrugs and/or pharmaceutically active ingredients. The presentlydisclosed and/or claimed inventive concept(s) may be used toencapsulate, attach, bind or otherwise be used to affect the storage,stability, longevity and/or release of any of the following drugs as thepharmaceutically active agent in a composition. One or more of thefollowing bioactive agents listed in (A)-(X) below may be combined withone or more carriers (however, said listing of agents provided in(A)-(X) is to be understood to be simply for illustration purposes, andis not to be construed as limiting):

(A) Analgesic anti-inflammatory agents such as, acetaminophen, aspirin,salicylic acid, methyl salicylate, choline salicylate, glycolsalicylate, 1-menthol, camphor, mefenamic acid, fluphenamic acid,indomethacin, diclofenac, alclofenac, ibuprofen, ketoprofen, naproxene,pranoprofen, fenoprofen, sulindac, fenbufen, clidanac, flurbiprofen,indoprofen, protizidic acid, fentiazac, tolmetin, tiaprofenic acid,bendazac, bufexamac, piroxicam, phenylbutazone, oxyphenbutazone,clofezone, pentazocine, mepirizole, and the like.

(B) Drugs having an action on the central nervous system, for examplesedatives, hypnotics, antianxiety agents, analgesics and anesthetics,such as, chloral, buprenorphine, naloxone, haloperidol, fluphenazine,pentobarbital, phenobarbital, secobarbital, amobarbital, cydobarbital,codeine, lidocaine, tetracaine, dyclonine, dibucaine, cocaine, procaine,mepivacaine, bupivacaine, etidocaine, prilocaine, benzocaine, fentanyl,nicotine, and the like. Local anesthetics such as, benzocaine, procaine,dibucaine, lidocaine, and the like.

(C) Antihistaminics or antiallergic agents such as, diphenhydramine,dimenhydrinate, perphenazine, triprolidine, pyrilamine, chlorcyclizine,promethazine, carbinoxamine, tripelennamine, brompheniramine,hydroxyzine, cyclizine, meclizine, clorprenaline, terfenadine,chlorpheniramine, and the like. Anti-allergenics such as, antazoline,methapyrilene, chlorpheniramine, pyrilamine, pheniramine, and the like.Decongestants such as, phenylephrine, ephedrine, naphazoline,tetrahydrozoline, and the like.

(D) Antipyretics such as, aspirin, salicylamide, non-steroidalanti-inflammatory agents, and the like. Antimigraine agents such as,dihydroergotamine, pizotyline, and the like. Acetonide anti-inflammatoryagents, such as hydrocortisone, cortisone, dexamethasone, fluocinolone,triamcinolone, medrysone, prednisolone, flurandrenolide, prednisone,halcinonide, methylprednisolone, fludrocortisone, corticosterone,paramethasone, betamethasone, ibuprophen, naproxen, fenoprofen,fenbufen, flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin,piroxicam, aspirin, salicylic acid, diflunisal, methyl salicylate,phenylbutazone, sulindac, mefenamic acid, meclofenamate sodium,tolmetin, and the like. Muscle relaxants such as, tolperisone, baclofen,dantrolene sodium, cyclobenzaprine.

(E) Steroids such as, androgenic steriods, such as, testosterone,methyltestosterone, fluoxymesterone, estrogens such as, conjugatedestrogens, esterified estrogens, estropipate, 17-β estradiol, 17-βestradiol valerate, equilin, mestranol, estrone, estriol, 17β ethinylestradiol, diethylstilbestrol, progestational agents, such as,progesterone, 19-norprogesterone, norethindrone, norethindrone acetate,melengestrol, chlormadinone, ethisterone, medroxyprogesterone acetate,hydroxyprogesterone caproate, ethynodiol diacetate, norethynodrel, 17-αhydroxyprogesterone, dydrogesterone, dimethisterone, ethinylestrenol,norgestrel, demegestone, promegestone, megestrol acetate, and the like.

(F) Respiratory agents such as, theophilline and β2-adrenergic agonists,such as, albuterol, terbutaline, metaproterenol, ritodrine, carbuterol,fenoterol, quinterenol, rimiterol, solmefamol, soterenol, tetroquinol,and the like. Sympathomimetics such as, dopamine, norepinephrine,phenylpropanolamine, phenylephrine, pseudoephedrine, amphetamine,propylhexedrine, arecoline, and the like.

(G) Antimicrobial agents including antibacterial agents, antifungalagents, antimycotic agents and antiviral agents; tetracyclines such as,oxytetracycline, penicillins, such as, ampicillin, cephalosporins suchas, cefalotin, aminoglycosides, such as, kanamycin, macrolides such as,erythromycin, chloramphenicol, iodides, nitrofrantoin, nystatin,amphotericin, fradiomycin, sulfonamides, purrolnitrin, clotrimazole,miconazole chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine,sulfamerazine, sulfamethizole and sulfisoxazole; antivirals, includingidoxuridine; clarithromycin; and other anti-infectives includingnitrofurazone, and the like.

(H) Antihypertensive agents such as, clonidine, α-methyldopa, reserpine,syrosingopine, rescinnamine, cinnarizine, hydrazine, prazosin, and thelike. Antihypertensive diuretics such as, chlorothiazide,hydrochlorothrazide, bendoflumethazide, trichlormethiazide, furosemide,tripamide, methylclothiazide, penfluzide, hydrothiazide, spironolactone,metolazone, and the like. Cardiotonics such as, digitalis,ubidecarenone, dopamine, and the like. Coronary vasodilators such as,organic nitrates such as, nitroglycerine, isosorbitol dinitrate,erythritol tetranitrate, and pentaerythritol tetranitrate, dipyridamole,dilazep, trapidil, trimetazidine, and the like. Vasoconstrictors suchas, dihydroergotamine, dihydroergotoxine, and the like. β-blockers orantiarrhythmic agents such as, timolol pindolol, propranolol, and thelike. Humoral agents such as, the prostaglandins, natural and synthetic,for example PGE1, PGE2α, and PGF2α, and the PGE1 analog misoprostol.Antispasmodics such as, atropine, methantheline, papaverine,cinnamedrine, methscopolamine, and the like.

(I) Calcium antagonists and other circulatory organ agents, such as,aptopril, diltiazem, nifedipine, nicardipine, verapamil, bencyclane,ifenprodil tartarate, molsidomine, clonidine, prazosin, and the like.Anti-convulsants such as, nitrazepam, meprobamate, phenytoin, and thelike. Agents for dizziness such as, isoprenaline, betahistine,scopolamine, and the like. Tranquilizers such as, reserprine,chlorpromazine, and antianxiety benzodiazepines such as, alprazolam,chlordiazepoxide, clorazeptate, halazepam, oxazepam, prazepam,clonazepam, flurazepam, triazolam, lorazepam, diazepam, and the like.

(J) Antipsychotics such as, phenothiazines including thiopropazate,chlorpromazine, triflupromazine, mesoridazine, piperracetazine,thioridazine, acetophenazine, fluphenazine, perphenazine,trifluoperazine, and other major tranqulizers such as, chlorprathixene,thiothixene, haloperidol, bromperidol, loxapine, and molindone, as wellas those agents used at lower doses in the treatment of nausea,vomiting, and the like.

(K) Drugs for Parkinson's disease, spasticity, and acute muscle spasmssuch as levodopa, carbidopa, amantadine, apomorphine, bromocriptine,selegiline (deprenyl), trihexyphenidyl hydrochloride, benztropinemesylate, procyclidine hydrochloride, baclofen, diazepam, dantrolene,and the like. Respiratory agents such as, codeine, ephedrine,isoproterenol, dextromethorphan, orciprenaline, ipratropium bromide,cromglycic acid, and the like. Non-steroidal hormones or antihormonessuch as, corticotropin, oxytocin, vasopressin, salivary hormone, thyroidhormone, adrenal hormone, kallikrein, insulin, oxendolone, and the like.

(L) Vitamins such as, vitamins A, B, C, D, E and K and derivativesthereof, calciferols, mecobalamin, and the like for dermatologicallyuse. Enzymes such as, lysozyme, urokinaze, and the like. Herbalmedicaments or crude extracts such as, Aloe vera, and the like.

(M) Antitumor agents such as, 5-fluorouracil and derivatives thereof,krestin, picibanil, ancitabine, cytarabine, and the like. Anti-estrogenor anti-hormone agents such as, tamoxifen or human chorionicgonadotropin, and the like. Miotics such as pilocarpine, and the like.

(N) Cholinergic agonists such as, choline, acetylcholine, methacholine,carbachol, bethanechol, pilocarpine, muscarine, arecoline, and the like.Antimuscarinic or muscarinic cholinergic blocking agents such as,atropine, scopolamine, homatropine, methscopolamine, homatropinemethylbromide, methantheline, cyclopentolate, tropicamide,propantheline, anisotropine, dicyclomine, eucatropine, and the like.

(O) Mydriatics such as, atropine, cyclopentolate, homatropine,scopolamine, tropicamide, eucatropine, hydroxyamphetamine, and the like.Psychic energizers such as 3-(2-aminopropy)indole,3-(2-aminobutyl)indole, and the like.

(P) Antidepressant drugs such as, isocarboxazid, phenelzine,tranylcypromine, imipramine, amitriptyline, trimipramine, doxepin,desipramine, nortriptyline, protriptyline, amoxapine, maprotiline,trazodone, and the like.

(Q) Anti-diabetics such as, insulin, and anticancer drugs such as,tamoxifen, methotrexate, and the like.

(R) Anorectic drugs such as, dextroamphetamine, methamphetamine,phenylpropanolamine, fenfluramine, diethylpropion, mazindol,phentermine, and the like.

(S) Anti-malarials such as, the 4-aminoquinolines, alphaaminoquinolines,chloroquine, pyrimethamine, and the like.

(T) Protein therapeutics such as enzymes, cytokines, growth factors,hormones, receptors, antibodies, immune complexes, and the like. Alsoincluded are protein derivatives that enhance or block the activity ofany of the naturally-occurring or isolated molecules listed herein orinteracting components in the biochemical or cellular pathways.

(U) Anti-ulcerative agents such as, misoprostol, omeprazole, enprostil,and the like. Antiulcer agents such as, allantoin, aldioxa, alcloxa,N-methylscopolamine methylsuflate, and the like. Antidiabetics such asinsulin, and the like.

(V) Anti-cancer agents such as, cis-platin, actinomycin D, doxorubicin,vincristine, vinblastine, etoposide, amsacrine, mitoxantrone,tenipaside, taxol, colchicine, cyclosporin A, phenothiazines orthioxantheres.

(W) For use with vaccines, one or more antigens, such as, natural,heat-killer, inactivated, synthetic, peptides and even T cell epitopes(e.g., GADE, DAGE, MAGE, etc.) and the like.

(X) Example therapeutic or active agents also include water soluble orpoorly soluble drugs of molecular weights from 40 to 1,100 including thefollowing: Hydrocodone, Lexapro, Vicodin, Effexor, Paxil, Wellbutrin,Bextra, Neurontin, Lipitor, Percocet, Oxycodone, Valium, Naproxen,Tramadol, Ambien, Oxycontin, Celebrex, Prednisone, Celexa, Ultracet,Protonix, Soma, Atenolol, Lisinopril, Lortab, Darvocet, Cipro, Levaquin,Ativan, Nexium, Cyclobenzaprine, Ultram, Alprazolam, Trazodone, Norvasc,Biaxin, Codeine, Clonazepam, Toprol, Zithromax, Diovan, Skelaxin,Klonopin, Lorazepam, Depakote, Diazepam, Albuterol, Topamax, Seroquel,Amoxicillin, Ritalin, Methadone, Augmentin, Zetia, Cephalexin, Prevacid,Flexeril, Synthroid, Promethazine, Phentermine, Metformin, Doxycycline,Aspirin, Remeron, Metoprolol, Amitriptyline, Advair, Ibuprofen,Hydrochlorothiazide, Crestor, Acetaminophen, Concerta, Clonidine, Norco,Elavil, Abilify, Risperdal, Mobic, Ranitidine, Lasix, Fluoxetine,Coumadin, Diclofenac, Hydroxyzine, Phenergan, Lamictal, Verapamil,Guaifenesin, Aciphex, Furosemide, Entex, Metronidazole, Carisoprodol,Propoxyphene, Digoxin, Zanaflex, Clindamycin, Trileptal, Buspar, Keflex,Bactrim, Dilantin, Flomax, Benicar, Baclofen, Endocet, Avelox, Lotrel,Inderal, Provigil, Zantac, Fentanyl, Premarin, Penicillin, Claritin,Reglan, Enalapril, Tricor, Methotrexate, Pravachol, Amiodarone, Zelnorm,Erythromycin, Tegretol, Omeprazole, and Meclizine.

The drugs mentioned above may be used in combination as required.Moreover, the above drugs may be used either in the free form or, ifcapable of forming salts, in the form of a salt with a suitable acid orbase. If the drugs have a carboxyl group, their esters may be employed.

The “suitable acid” may be an organic acid, for example, methanesulfonicacid, lactic acid, tartaric acid, fumaric acid, maleic acid, aceticacid, or an inorganic acid, for example, hydrochloric acid, hydrobromicacid, phosphoric acid or sulfuric acid. The base may be an organic base,for example, ammonia, triethylamine, or an inorganic base, for example,sodium hydroxide or potassium hydroxide. The esters may be alkyl esters,aryl esters, aralkyl esters, and. the like.

Bioactive Delivery of the Heparosan Conjugate

The heparosan conjugate may be administered parenterally,intraperitoneally, intraspinally, intravenously, intramuscularly,intravaginally, subcutaneously, intranasally, rectally, orintracerebrally. Dispersions of the heparosan conjugate may be preparedin glycerol, liquid poly[ethylene glycols], and mixtures thereof, aswell as in oils. Under ordinary conditions of storage and use, suchpreparations of the heparosan conjugate may also contain a preservativeto prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injection use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The heparosan conjugate may be used in conjunctionwith a solvent or dispersion medium containing, for example, water,ethanol, poly-ol (for example, glycerol, propylene glycol, and liquidpoly[ethylene glycol], and the like), suitable mixtures thereof,vegetable oils, and combinations thereof.

The proper fluidity of the heparosan conjugate may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion, and/or by the useof surfactants. Prevention of the action of microorganisms may beachieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars, sodium chloride, or polyalcohols such as mannitoland sorbitol, in the composition. Prolonged absorption of the injectablecompositions may be brought about by including in the composition anagent that delays absorption, for example, aluminum monostearate orgelatin.

Sterile injectable solutions may be prepared by incorporating theheparosan conjugate in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the heparosan conjugate into a sterile carrier thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationmay include vacuum drying, spray drying, spray freezing andfreeze-drying that yields a powder of the active ingredient (i.e., theheparosan conjugate) plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The heparosan conjugate may be orally administered, for example, with aninert diluent or an assimilable edible carrier. The heparosan conjugateand other ingredients may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe subject's diet. For oral therapeutic administration, the heparosanconjugate may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of theheparosan conjugate in the compositions and preparations may, of course,be varied as will be known to the skilled artisan. The amount of theheparosan conjugate in such therapeutically useful compositions is suchthat a suitable dosage will be obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of heparosan conjugate calculated toproduce the desired therapeutic effect. The specification for the dosageunit forms of the presently disclosed and/or claimed inventiveconcept(s) are dictated by and directly dependent on (a) the uniquecharacteristics of the heparosan conjugate and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a subject.

Aqueous compositions of the present invention comprise an effectiveamount of the nanoparticle, nanofibril or nanoshell or chemicalcomposition of the presently disclosed and/or claimed inventiveconcept(s) dissolved and/or dispersed in a pharmaceutically acceptablecarrier and/or aqueous medium. The biological material should beextensively dialyzed to remove undesired small molecular weightmolecules and/or lyophilized for more ready formulation into a desiredvehicle, where appropriate. The active compounds may generally beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, sub-cutaneous, intralesional, and/oreven intraperitoneal routes. The preparation of an aqueous compositionthat contains an effective amount of the nanoshell composition as anactive component and/or ingredient will be known to those of skill inthe art in light of the present disclosure. Typically, such compositionsmay be prepared as injectables, either as liquid solutions and/orsuspensions; solid forms suitable for using to prepare solutions and/orsuspensions upon the addition of a liquid prior to injection may also beprepared; and/or the preparations may also be emulsified. Also, theheparosan vehicle can be used to enhance a secondary vehicle (e.g.,liposomes, nanoparticles, etc.) that acts as a carrier or adjuvant for adrug.

Examples are provided hereinbelow. However, the present invention is tobe understood to not be limited in its application to the specificexperimentation, results and laboratory procedures. Rather, the Examplesare simply provided as one of various embodiments and is meant to beexemplary, not exhaustive.

EXAMPLE 1

Defined GAG synthesis and heparosan synthesis in particular is ratherversatile with respect to chemical functionality as well as sizecontrol. For example, U.S. Publication No. US 2008/0109236 A1 (U.S.patent application Ser. No. 11/906,704 filed Oct. 3, 2007, entitled“PRODUCTION OF DEFINED MONODISPERSE HEPAROSAN POLYMERS AND UNNATURALPOLYMERS WITH POLYSACCHARIDE SYNTHASES”) discloses a methodology forpolymer grafting utilizing heparin/heparosan synthases from Pasteurellain order to provide heparosan polymers having a targeted size and thatare substantially monodisperse at the desired size ranges. As disclosedin the '704 application, appropriate reactive moieties may be added tothe heparosan polymer at the reducing or non-reducing termini orthroughout the sugar chain. Having one reactive group/chain ispreferable when conjugating the heparosan polymer to its cargo. As such,the methodology of the '704 application can be applied to produceheparosan polymers suitable for HEPylation with a cargo molecule. Table4 lists different HEPylation polymer chemistries which are availableand/or suitable for modifying the heparosan polymer to make it moreacceptable or suitable for conjugating with specific cargo molecules. Itis not the nature or manner of the complexation or conjugation betweenheparosan and the drug (by any covalent chemical or weak bond) that iscontrolling; rather, it is the particular use to which the heparosanwill be put.

TABLE 4 Heparosan Polymer Chemistries Available Functional Number ofReactive Position on Typical Group Extra Steps * with: Heparosan CargoNotes 1. aldehyde 0 amines Reducing Peptide, Irreversible if ProteinNaCNBH₃ coupling 2. malemide 2 sulfhydryls Reducing Peptide,Irreversible Protein 3. pyridylthio 2 sulfhydryls Reducing Peptide,Reversible (disulfide) Protein 4. Azido 1 acetylenes Non- Various Cu(I)Coupling (tripleCC Reducing Irreversible bond) Interior 5. Amino 1aldehydes Reducing Drugs NaCNBH₄ Coupling Non- Irreversible reducingInterior 6. N-hydroxy 2 amines Reducing Peptide, Irreversiblesuccimimide Non- Protein (NHS) reducing 7. hydrazide 1 aldehydes,Reducing Drugs Irreversible if ketones NaCNBH₃ Reversible otherwise *beyond “normal heparosan” polymer

PmHS1 (SEQ ID NOS: 1 and 2, the amino acid and nucleotide sequences,respectively) was expressed as a carboxyl terminal fusion to maltosebinding protein (MBP) using the pMAL-c2X vector (New England BioLabs).To facilitate extracting the enzymes, the expression host E. coli XJa(Zymo Research), which encodes a phage lysin enzyme, was employed andallowed for simple freeze/thaw lysis. Cultures were grown in SuperiorBroth (AthenaES) at 30° C. with ampicillin (100 μg/ml), and L-arabinose(3.25 mM). At mid-log phase, isopropyl β-D-1-thiogalactopyranoside(IPTG) (0.2 mM final) was added to induce fusion protein production. Onehour after induction, the cultures were supplemented with fructose (12.8mM final) and grown for approximately 5-12 hours before harvesting bycentrifugation at 4° C. The bacteria were resuspended in 20 mM Tris, pH7.2, and protease inhibitor cocktail on ice, then frozen and thawedtwice, thus allowing lysin to degrade the cell walls. The lysates wereclarified by centrifugation.

The synthase was affinity purified via the MBP unit using amylose resin(New England BioLabs). After washing extensively with column buffer (20mM Tris, pH 7.2, 200 mM NaCl, 1 mM EDTA), the protein was eluted incolumn buffer containing 10 mM maltose. Protein concentration wasquantitated by the Bradford assay (Pierce, Rockford, Ill.) using abovine albumin serum standard. The purification was monitored bySDS-PAGE with copper negative staining (which adds comparablesensitivity as conventional silver staining) followed by Coomassie bluestaining. The enzyme (approximately 90-95% pure; yield ˜10 mg per literof culture) may be used directly after buffer exchange into 50 mM Tris,pH 7.2, by ultrafiltration. Further purification by anion-exchangechromatography provides an approximately 95-99% pure PmHS1 enzyme.

A heparosan polysaccharide (having a molecular weight of approximately200-300 kDa) derived from the spent fermentation broth of P. multocidaType D cultures was converted into heparosan tetrasaccharide (4-mer,having a molecular weight of approximately 700 Da), the startingmaterial for the primers described later herein. P. multocida Type Dcells were grown in a proprietary synthetic media at 37° C. in shakeflasks for approximately 24 hrs. Spent culture medium (the liquid partof culture after microbial cells were removed) was harvested (bycentrifugation at 10,000×g, 20 min) and deproteinized (solventextraction with chloroform). The very large anionic heparosan polymer(“fermentation heparosan,” having a molecular weight of approximately200-300 kDa) was isolated via ultrafiltration (30 kDa molecular weightcut-off; Amicon) and ion exchange chromatography (NaCl gradient onQ-Sepharose; Pharmacia), Heparosan from E. coli K5 cultures can also beused, but the polymers are initially lower molecular weight than P.multocida Type D.

Heparosan oligosaccharides ((GIcUA-GlcNAc)_(n)-(GlcUA-anhydromannitol),n=1, 2 or 3) were prepared by partial deacetylation of heparosanpolysaccharide with base, nitrous acid hydrolysis, and reduction; thesepolymers contain intact non-reducing termini, but an anhydromannitolgroup at the reducing end. The fragments were purified by gel filtrationon a P2 column (BioRad, Hercules, Calif.) in 0.2 M ammonium formate,followed by normal phase thin layer chromatography (TLC) on silicaplates (Whatman) with n-butanol/acetic acid/water (1:1:1). The bandswere detected by staining of side lanes with napthoresorcinol. The sizeand purity of oligosaccharides were verified by matrix assisted laserdesorption ionization time of flight mass spectrometry (MALI-ToF MS).Alternatively, acid hydrolysis or enzymatic cleavage yieldsoligosaccharides that can also be employed for use.

Amino-Hep4 was prepared by reductive amination of Hep4, the heparosantetrasaccharide (n=2), with ammonium ion. The dry sugar was dissolved inanhydrous methanol (0.71 mg/ml w/v or 0.93 mM final) under sonication.After addition of solid ammonium acetate and NaBH₃CN (final 1 M and 0.1M, respectively), the mixture was heated to reflux (approximately 70-80°C.) overnight. Thin layer chromatographic analysis (TLC—silica;BuOH/AcOH/H₂O 1:1:1 v:v:v with detection by napthoresorcinol reagent)was used to monitor consumption of the starting material. The reactionwas quenched by slow addition of 20% AcOH. The solvent was evaporated invacuo and the residue dissolved in 0.2 M ammonium formate for desaltingby gel filtration chromatography on a P-2 resin column (Bio-Rad) in thesame volatile buffer. The fractions containing the target molecule werepooled and lyophilized. The volatile salts were removed by two morecycles of dissolving in water and lyophilization. Flash silica gelcolumn chromatography (silica gel 60, E. Merck; BuOH/AcOH/H₂O 1:1:1v:v:v) was employed for further purification. The structure of theamino-Hep₄ product was confirmed by matrix-assisted laser desorptiontime-of-flight mass spectrometry analysis. The derived amino-HEP4 primerwas extended by the PmHS1 enzyme as described in the '704 application toform amino-heparosan polymer used as the carrier portion of theheparosan conjugate.

The Amino-heparosan polymer may be further reacted with variousactivated bifunctional N-hydroxysuccinimide esters to thereby adddesirable groups including maleimides, a sulfhydryl selective reagent,etc. The amino-heparosan polymer was reacted with approximately 10-foldmolar excess of (Pierce) in 10% dimethylsulfoxide, 0.1 M potassiumphosphate, 0.15 M NaCl, pH 7.4, for 2 hrs at room temperature. Thetarget compound in the reaction mixture was purified by gel filtrationchromatography on Sephadex G-25 resin (PD-10, Pharmacia) as detailedabove.

The amino-HEP4 products may also be reductively aminated by treatmentwith adipic acid dihydrazide (30 eq) and sodium cyanoborohydride (100eq) at 50-60° C. In 1 M sodium phosphate buffer, pH 5.5. Afterdesalting, the obtained hydrazide amino-HEP4 may be further purified bystrong anion exchange chromatography using Sepharose Q (Pharmacia) withan ammonium bicarbonate gradient elution. The hydrazide amino-HEP4primer may also be extended by the PmHS1 enzyme in order to producepolymers having varying sizes as described below.

Synchronized polymerization reactions were used to produce monodispersepolymers as previously described and disclosed in the '704 patentapplication. The formation of heparosan with narrow size distribution(i.e., monodisperse) is dependent on the ability of the PmHS1 enzyme tobe primed by acceptors (thus avoiding a slow de novo initiation eventyielding out of step elongation events) and efficiently transfermonosaccharides from UDP-sugars. Recombinant PmHS1 synthesizes heparosanchains in vitro if supplied with both required UDP-sugars according tothe equation:

n UDP-GlcUA+n UDP-GlcNAc→2n UDP+[GlcUA-GlcNAc]_(n)

However, if a heparosan-like oligosaccharide ([GlcUA-GlcNAc]_(x)) isalso supplied in vitro, then the overall incorporation rate is elevatedup to approximately 25-fold. The rate of initiation of a new chain denovo is slower than the subsequent elongation (i.e., repetitive additionof sugars to a nascent HA molecule). The observed stimulation ofsynthesis by exogenous acceptor primer appears to operate by bypassingthe kinetically slower initiation step, allowing the elongation reactionto predominate as in the following equation:

n UDP-GlcUA+n UDP-GlcNAc+[GlcUA-GlcNAc]_(x)→2n UDP+[GlcUA-GlcNAc]_(x+n)

If there are many termini (i.e., z is large), then a limited amount ofUDP-sugars will be distributed among many molecules and thus result inmany short polymer chain extensions. Conversely, if there are fewtermini (i.e., z is small), then the limited amount of UDP-sugars willbe distributed among few molecules and thus result in long polymer chainextensions. Thus, by controlling the molar ratio of acceptor toUP-sugar, it is possible to select the final polymer size desired.Typically, from about 50% to about 90% of the starting UDP-sugars areconsumed in the reactions on the basis of polysaccharide recovery.Alternatively, if size control is not as critical, then “fermentationheparosan” or its fragments (generated by acid, base, enzyme or physicalcleavage methods known to those of skill in the art) will suffice as thevehicle. Similarly, chemically manufactured heparosan may be utilized.As will be appreciated by one of ordinary skill in the art, therefore,it is not the source or manner in which the heparosan is made that iscontrolling; rather, it is the particular use to which the heparosanwill be put. If size is critical, recombinant chemoenzymatic productionis preferred. In situations where size is of a secondary or lesserimportance, fermentation heparosan (or its derivatives) may be used. Assuch, the use of heparosan from any source or produced by anymethodology is intended to be within the presently claimed and disclosedinvention. Likewise, it is not the nature or manner of the complexationbetween heparosan and the drug (by any chemical or weak bond) that iscontrolling; rather it is the particular use to which the heparosan willbe put.

The yield and molecular weight size distribution of the heparosan ischecked by (a) carbazole assays for uronic acid; and (b) agarose gelelectrophoresis (1X TAE buffer, 0.8-1.5% agarose) followed by Stains-Alldetection. The carbazole assay is a spectrophotometric chemical assaythat measures the amount of uronic acid in the sample via production ofa pink color; every other sugar in the heparosan chain is a glucuronicacid (GlcUA). The heparosan polymer size is determined by comparison tomonodisperse HA size standards (HA Lo-Ladder, Hyalose, LLC) run on gels.The detection limit of the carbazole and the gel assays is approximately5-15 micrograms of polymer. Any endotoxin is removed by passage throughan immobilized polymyxin column (Pierce); the material is then testedwith a Limulus amoebacyte-based assay (www.Cambrex.com) to assure thatthe heparosan contains <0.05 endotoxin units/mg solid (based on USPguidelines).

Examples of the productions of monodisperse heparosan are shown in FIG.5 where providing various levels of primer yielded different Mw (weightaverage molecular mass) products with low polydispersity (Mw/Mn;Mn=number average molecular weight). For reference, the polydispersityvalue for an ideal monodisperse polymer equals 1. The parallel reactionwithout an acceptor (lane 0) resulted in a large product that wassignificantly polydisperse, i.e., it contains heparosan polymers ofvarying size and length.

The polymerization by synthases in the presence of an acceptor is asynchronized process. Reactions without acceptor exhibit a lag periodinterspersed with numerous, out of step initiation events that yield ashort heparosan oligosaccharide. Once any chain is formed, the heparosanpolymer is elongated rapidly. Other new chains that arise later duringthe lag period are also elongated rapidly, but the size of these youngerchains never catches up to the older chains in a reaction with a finiteamount of UDP-sugars. In contrast, in reactions containing an acceptor,all heparosan chains are elongated in parallel in a nonprocessivefashion resulting in a more homogenous final polymer population.

The enzymological properties of recombinant pmHS1 described above alsoallow for the control of heparosan polymer size in chemoenzymaticsyntheses. First, as noted above, the rate-limiting step in vitroappears to be the chain initiation step. Therefore, PmHS1 transfersmonosaccharides onto the existing heparosan acceptor chains beforesubstantial de novo synthesis. Second, the enzyme polymerizes heparosanin a rapid nonprocessive fashion in vitro. Therefore, the amount ofprimer should affect the final size of the product when a finite amountof UDP-sugar is present. The synthase adds all available UDP-sugarprecursors to the nonreducing. termini of acceptors as in the equation:

n UDP-GlcUA+n UDP-GlcNAc+z[GlcUA-GlcNAc]_(x)→2nUDP+z[GlcUA-GlcNAc]_(x+(n/z))

Thus, by controlling the molar ratio of acceptor to UDP-sugar, it is nowpossible to select the final heparosan polymer size desired. Typically,from about 50% to about 90% of the starting UDP-sugars are consumed inthe reactions on the basis of polysaccharide recovery.

The size distribution of the heparosan polymers produced was determinedby high performance size exclusion chromatography-multi angle laserlight scattering (SEC-MALLS). Polymers (2.5 to 12 μg mass; 50 μlinjection) were separated on PL aquagel—OH 30 (8 μm), —OH 40, —OH 50,—OH 60 (15 μm) columns (7.5×300 mm, Polymer Laboratories) in tandem oralone as required by the size range of the polymers to be analyzed. Thecolumns were eluted with 50 mM sodium phosphate, 150 mM NaCl, pH 7 at0.5 ml/min. MALLS analysis of the eluant was performed by a DAWN DSPLaser Photometer in series with an OPTILAB DSP InterferometricRefractometer (632.8 nm; Wyatt Technology). The ASTRA software packagewas used to determine the absolute average molecular mass using a dn/dccoefficient of 0.153 determined for HA, a polymer with the exact samesugar composition as heparosan, by Wyatt Technology. The Mw andpolydispersity values from at least two SEC-MALLS runs were averaged inorder to obtain a final approximation of the Mw and polydispersity ofthe heparosan molecule.

Although there have been described many different types of moleculesthat can be conjugated with the heparosan polymer, two primary definedmodel cargoes are of particular interest and importance: (a) achemotherapy agent, and (b) a protein therapeutic. For (a), doxorubicinand taxol are useful chemotherapy agents for treating several cancers.Taxol is only slightly soluble in water (i.e., approximately 0.4micrograms per mL), and such solubility issues can be improved throughconjugation with the hydrophilic heparosan polymer. The carbonyl groupsfound on the taxol or doxorubicin molecule allows the drug to couplemonovalently to the heparosan polymer, thereby providing a heparosandrug conjugate. However, if desirable and to increase dosage ofpharmaceutical available for pharmocological treatment, the drugmolecule is also attachable to multiple positions on the heparosanpolymer. A dihydrazide may also be added to the drug-heparosan conjugatein order to create a time-release formulation. The heparosan-doxorubicinor taxol-heparosan conjugate is water-soluble and nontoxic; as heparosanis slowly degraded in blood pH, the linkage releases free activedoxorubicin or taxol in a specific and controlled manner.

For (b), protein targets include enzymes, cytokines, interferon,antibodies, receptors and growth factors as well as modified derivatives(e.g., with either chemical or molecular genetic changes). Bovine serumalbumin, BSA, is a useful surrogate for testing and modeling a proteintherapeutic conjugated with heparosan. The BSA protein does not haveintrinsic glycosylation and facilitates analysis of the addition of oneor more heparosan chains to the BSA-heparosan conjugate. The use ofheparosan as vehicle for drug conjugation is also applicable torecombinant proteins with a bioengineered extra cysteine or an exposedsulfhydryl group, such as antibodies, resulting in an improved strategyto couple such cargo. The cysteine's sulfhydryl group is coupled to themonovalent heparosan-maleimide to provide the heparosan conjugate.Alternatively, heparosan-thiopyridyl may be used if protein release isdesirable due to a reversible disulfide linkage between the sugarpolymer (i.e., the heparosan polymer) and the cargo (i.e., recombinantproteins, etc.). As with PEG, the aldehyde of heparosan chains may becoupled to amines of proteins via reductive amination with sodiumcyanoborohydride; a useful process for the conjugation of growth factorsand interferon.

After the heparosan protein conjugation occurs, the molecule ischallenged with (a) proteases and (b) antibodies (e.g., anti-BSAantibody). For protease sensitivity, samples of protein orprotein-heparosan are treated with dilution series of trypsin, anaggressive serine protease, for 0-60 min, then run on the SDS-PAGE gel.Relative resistance to digestion occurs for protein-heparosan. Forantigenicity, protein or protein-heparosan are incubated withanti-protein IgG beads (e.g., anti-BSA IgG beads from Sigma) for 1 hourin saline, then the supernatant analyzed by PAGE. Alternatively, solubleanti-protein reagent can be incubated with test samples and run onnative gels (similar to standard gels except that sample buffer lacksreducing agent and will not be boiled). A higher molecular weightcomplex forms when the antibody builds to the protein-heparosanconjugate causing a “super-shift”. The protein-heparosan conjugate isresistant to anti-protein if the heparosan blocks its epitope. Theoverall goal is to assess, and to optimize, as needed the reactionparameters to produce the heparosan-conjugate.

A Strategy for HEPylation Reagent Preparation and Utilization

Three or four sequential reactions were used to produce a HEPylatedcargo in this embodiment of drug-conjugate synthesis as in FIG. 6. (1)An acceptor (a heparosan tetrasaccharide, Hep4) was modified to add areactive group (R=amino or hydrazide). (2) Three independent reactionmixtures, each with a different ratio of UDP-sugar/reactive acceptor,were elongated with PmHS1 synthase to yield a set of distinct reactivemonodisperse heparosan polymer preparations of three different sizes.(3) The Cargo was modified directly with any one size polymer. (3A)Alternatively (dotted line), an additional modification step was used toalter the reactivity of the heparosan reagent (add a R′=maleimide grouponto amino-heparosan) allowing the next step, (4), modification of thecargo.

There are numerous possibilities for coupling heparosan vehicle andtherapeutic cargo involving various chemistries which include, but arenot limited to, the examples listed previously in Table 4. In theexamples that follow, two proteins, BSA and IgG antibody, and two smallmolecules, fluorescein and Bolton-Hunter reagent, were used as cargo forcoupling various monodisperse or polydisperse heparosan polymersproduced either via fermentation in vivo or chemoenzymatic synthesis invivo.

FIGS. 7-8 illustrate the production of HEPylated BSA molecules viachemoenzymatic synthesis. In FIG. 7 radioactive bovine serum albumin[BSA] (¹²⁵I-Bolton-Hunter labeled; migration marked with arrow) proteinwas reacted via reductive amination with sodium cyanoborohydride withtwo different reactive 20 kDa heparosan polysaccharides, ‘H’ or ‘N’(unmodified BSA starting material is lane ‘0’). Each reactive heparosanwas made by extending a short oligosaccharide acceptor into a longer 20kDa polymer with PmHS1 enzyme and UDP-sugars. The acceptors were derivedfrom heparosan polysaccharide (˜200-300 kDa) by two different methods:for H, a heparosan tetrasaccharide formed by HCl cleavage with generalstructure [GlcUA-GlcNAc]₂ was used while for N, a heparosantetrasaccharide formed by base treatment followed by nitrous acidcleavage with general structure [GlcUA-GlcNAc]-GlcUA-anhydromannitol wasused. As seen by the SDS-PAGE gel visualized by autoradiography on theleft, higher molecular weight products are observed in the N lane,corresponding to a series of HEPylated BSA molecules (see bracketedarea). The H lane does not have the same pattern due to the fact thatthe H polymer must mutarotate to yield a free aldehyde that can reactwith the BSA amine groups, and thus has lower yields. On the other hand,the N polymer always has a free aldehyde, thus allowing better reaction.As a proof of the HEPylated BSA structure, a duplicate sample of the Nmaterial was treated with heparosan lyase, an enzyme that degrades theheparosan polymer but does not degrade other macromolecules such as BSA.As seen in the ‘+ lyase lane,’ the BSA now runs at its originalposition, demonstrating that authentic heparosan chains were added tothe protein cargo.

In FIG. 8, bovine serum albumin [BSA] protein was reacted with Traut'sreagent (T; iminothiolane) to convert some of its amino groups (lysinesand amino termini) into free sulfhydryl groups forming T-BSA; in thiscase, ˜1-3 residues on average were predicted to be modified based onthe reaction stoichiometry employed and the general completeness of thereaction. This T-BSA material was incubated with a reactive 75 kDamaleimide heparosan. The reactive heparosan was made by (i) converting aheparosan tetrasaccharide ([GlcUA-GlcNAc]-GlcUA-anhydromannitol) into anamino derivative using reductive amination with sodium cyanoborohydridein the presence of ammonia, (ii) extending this sugar into a longerpolymer with PmHS1 enzyme and UDP-sugars, and (ii) reacting the longamino-polymer with a N-hydroxysuccinimide ester of amaleimide-containing. compound. As seen by the SDS-PAGE gel visualizedby Coomassie staining, a series of higher molecular weight products areobserved in the ‘Hep’ lane corresponding to a series of HEPylated BSAmolecules (untreated T-BSA control is in lane 0). The gel filtrationchromatography profile (with absorbance at 280 nm detection) confirmsthat higher molecular weight polymers, HEPylated cargo, were formed. Theproduction of several species is due to the nature of the chemicallymodified T-BSA (˜1 to 3 T reagents/BSA molecule are formed in a ratheruncontrolled chemical reaction); if a natural protein or a geneticallyengineered molecule (e.g., with an extra free cysteine residue)contained only a single sulfhydryl group, then a single mono-HEPylatedspecies would result. In addition, in other embodiments, any sulfhydrylmoiety could be used on the cargo (protein or other small molecule orsecondary vehicles) as well as any alternative sulfhydryl-reactivereagent on the heparosan polymer including pyridylthiols or haloacids.

Immunoreactivity of many drugs is a serious issue, thus necessitatingconjugation or humanization of the drug or the use of very low dosagesand/or short treatments. If heparosan is attached to a therapeuticcargo, then it is expected that the cargo surface will be lessaccessible to antibody binding. The HEPylated BSA material (BSA with oneto three 75 kDa heparosan chains/polypeptide) produced in experimentsdepicted in FIG. 8 was subjected to tests with an anti-BSA polyclonalantibody. To test this hypothesis, the FIG. 8 material (either di-,tri-, or mono-HEPylated BSA—all purified by gel filtration) werecompared to BSA and T-BSA (controls) in a radiometric immune assay (RIA)in a competition format (Table 5). First, a capture antibody coating wasplaced on the surface of a well of a 96-well plate. After blocking withovalbumin, a solution of one of the 4 test molecules above was added tothe wells together with radioactive BSA ([¹²⁵I] Bolton-Hunter labeled).After extensive washing, the wells were counted to measureradioactivity. Each competitor protein was measured at twoconcentrations, 25 or 500 nanograms (ng). The control wells did not haveany BSA competitor, thus representing a ‘maximal binding’ signal. BSAalone competed for binding with the radioactive probe to immobilizedantibody; the signal was substantially reduced by 25 ng of BSAcompetitor and greatly reduced with 500 nanograms of BSA. T-BSA withoutheparosan competed in a fashion similar to normal BSA. However, moreHEPylated BSA was needed to partially inhibit the radioactive signal,indicating that the antibody did not recognize or bind to the HEPylatedmolecules as well as BSA or T-BSA. Therefore, HEPylation will helpshield cargo from the full brunt of immunological defenses in themammalian body.

TABLE 5 Radiometric Immune Assay (RIA) comparing HEPylated BSA to BSAand T-BSA Sample [¹²⁵I] cpm Bound No competitor 1190 1270 BSA, 25 ng 750760 BSA, 500 ng 140 100 T-BSA, 25 ng 770 830 T-BSA, 500 ng 140 190 Di,tri-HEP BSA, 25 ng 1050 Di, tri-HEP BSA, 500 ng 620 mono-HEP BSA, 25 ng1220 mono-HEP BSA, 500 ng 630

FIG. 9 illustrates the production of HEPylated IgG molecules. Apreparation of radioactive immunoglobulins [IgG] (¹²⁵I-Bolton-Hunterlabeled; migration marked with bracket) was oxidized with sodiumperiodate to create new aldehydes on the IgG sugar moieties on the Fcregion. This oxidized glycoprotein was reacted via reductive aminationwith reactive hydrazide heparosan polysaccharide using sodiumcyanoborohydride. For preparation of reactive heparosan, a shortheparosan tetrasaccharide acceptor (derived from nitrous acid asdescribed earlier) was first treated with adipic dihydrazide (a compoundwith 2 terminal hydrazide functional groups; one end couples with sugarand the other end remains free for reaction with cargo) and thenextended into a longer ˜20 kDa polymer with PmHS1 enzyme and UDP-sugars.As seen by the SDS-PAGE gel visualized by autoradiography, highermolecular weight products were observed in the ‘Hep’ lane, correspondingto HEPylated IgG molecules (see arrow area) (unmodified IgG startingmaterial is lane ‘0’; note that IgG preparations from serum containmultiple species due to the variety of heavy and light chains and thereis also a small amount of higher molecular weight contaminant). Thisdata demonstrates yet another chemistry for coupling heparosan vehicleto a therapeutic cargo such as a glycoprotein like an antibody.

FIG. 10 illustrates the production of HEPylated fluorescein molecules.To produce these molecules, a preparation of reactive hydrazide 75 kDaheparosan (similar reagent as in FIG. 9) was reacted with fluoresceinisothiocyanate (FITC). As a control, heparosan without the reactivehydrazide group was also treated with the same FITC reagent (lane ‘0’).As seen by the PAGE gel visualized by virtue of ultraviolet-inducedfluorescence, a higher molecular weight fluorescent product was observedin the ‘Hy’ lane, corresponding to HEPylated fluorescein molecules (seearrow) (unreacted FITC starting material is bracketed). This datademonstrates yet another example of coupling heparosan vehicle to asmall molecule that is a proxy for a therapeutic cargo. In this case,the hydrazide linkage is meta-stable at physiological pH thus theHEPylated cargo will break down over time, facilitating time-releasedelivery of free small molecule. In the case of certain toxictherapeutics such as cancer chemotherapy drugs, this is a useful dosingfeature.

Alternatively, the cargo can first be coupled to the reactive acceptor,and then the heparosan chain added by polymer grafting with PmHS1 (e.g.,elongate the acceptor while coupled to cargo) due to the mild reactionconditions as shown in the example of FIG. 11. In FIG. 12, radioactivebovine serum albumin (²⁵I-Bolton-Hunter labeled BSA) protein was reactedvia reductive amination with sodium cyanoborohydride with two differentreactive oligosaccharide acceptors derived from heparosan (sameacceptors as in FIG. 7). For H, a heparosan tetrasaccharide formed byHCl cleavage with general structure [GlcUA-GlcNAc]₂ was used while forN, a heparosan tetrasaccharide formed by base treatment followed bynitrous acid cleavage with general structure[GlcUA-GlcNAc]-GlcUA-anhydromannitol was used. Then the shortoligosaccharide acceptor covalently attached onto the BSA was extendedvia polymer grafting into a longer heparosan polymer with PmHS1 enzymeand UDP-sugars. As seen by the SDS-PAGE gel visualized byautoradiography on the left, higher molecular weight products wereobserved in the N lane corresponding to HEPylated BSA molecules (seebracketed area; the unmodified BSA starting material is lane ‘0’ and ismarked with arrow).

Therefore, two different strategies can be used to add the heparosanvehicle onto the therapeutic cargo: (1) a long reactive polymer isdirectly added to the cargo as shown in the schematic model of FIG. 6and the data in FIG. 7 or (II) a short reactive acceptor is directlyadded to the cargo and then this conjugated molecule is elongated viapolymer grafting with PmHS1 and UDP-sugars as in FIG. 12 (data shownhere on the left and schematic model on the right).

In another example, heparosan produced by bacteria in vivo can bepurified and (a) coupled via its reducing end aldehyde or (b) activatedto couple to cargo (this latter approach with fermentation-derivedheparosan results in functional, but more heterogeneous final productswith higher polydispersity). In FIG. 13, radioactive bovine serumalbumin (¹²⁵I-Bolton-Hunter labeled BSA; migration marked with arrow;lane 0-=no treatment; note that some contaminating minor bands are alsopresent in all lanes) protein was reacted via reductive amination withsodium cyanoborohydride with periodate-oxidized reactive (contains newaldehyde groups) ˜200 kDa heparosan polysaccharide formed byfermentation of Pasteurella multocida Type F bacteria in vivo. Severalreaction pHs from pH 5-9 were tested. As seen by the SDS-PAGE gelvisualized by autoradiography, higher molecular weight product wasobserved in the reaction lanes (pH 5, 7.2, or 9) corresponding toHEPylated BSA molecules (see bracketed area). Therefore, in addition tochemoenzymatic synthetically derived heparosan vehicles, naturallyoccurring heparosan from microbes may also be used as the source ofvehicle. Such polymers include, but are not limited to, recombinantmicrobial hosts (e.g., Escherichia, Bacillus) with heparosan synthasessuch as PmHS1 or other native microbes that produce heparosan such asEscherichia coli K5.

In order to monitor the half-life (t1/2) and persistence of heparosan ina mouse or rat model, radioactive probes have been employed; however,ELISA or NMR may also be used to monitor the cargo. Typical elongationreactions contain: 50 mM Tris, pH 7.2, 1 mM MnCl₂, 1 to 50 mMUDP-sugars, 0.1 mg/ml PmHS1 enzyme and a primer; the stoichiometricratio of primer to UDP-sugars controls the heparosan chain size.

¹²⁵I-Heparosan: A heparosan oligosaccharide primer with a¹²⁵I-Bolton-Hunter reagent (a proxy for the therapeutic cargo) waselongated to any desired length with the PmHS1 enzyme. A series ofpolymers having distinct sizes, but equal radiochemical specificactivity (approximately 70 Ci/mmol) were generated; an example of onesuch radioactive conjugate is depicted in FIG. 14. The radioactivetetrasaccharide primer was made from heparosan polysaccharide by partialde-acetylation in base, nitrous acid cleavage, reductive amination withammonia, then coupling to NHS ester of ¹²⁵I-Bolton-Hunter reagent(Perkin Elmer NEN). The strong gamma-rays were readily detectable insamples of blood or tissues without additional processing. A series of¹²⁵I-heparosan probes were created to monitor the activity andfunctionality of the heparosan conjugate. Here, the rat was used as themodel to track the fate of the heparosan conjugate after injection, butother mammals such as man are expected to behave similarly.

Three commonly employed modes of a therapeutic injection were tested:(a) intravenous (i.v.); (b) intraperitoneal (i.p.) and (c) intramuscular(i.m). On the order of 5-50 uCi (approximately 10-100×10⁶ dpm)radioactive heparosan probe in a small volume of saline per mouse or ratwas injected at time zero into the tail vein or an implanted jugularport (i.v.) or abdominal cavity (i.p.) or the rear flank (i.m.). A groupof animals was injected in parallel. The mice or rats were kept inspecial cages to facilitate urine and feces collection. At various times(typically 5, 30, 60, 120, minutes and 1 to 5 days), blood was drawnfrom the animals. Duplicate animals were used for each time point. Someanimals were sacrificed at early time points for harvesting organs thatare known for potentially interacting with injected therapeutics (e.g.,the kidney and the liver). The fate of injected heparosan conjugate inthe mammalian body is shown in the example of FIG. 3.

For ¹²⁵I-probes, the samples (equal volumes or weights per sample) wereplaced directly in test tubes and measured with a solid-state gammascintillation counter for 1 minute. For detecting low amounts ofradioactivity, the counting interval was extended to 5 or 10 minutes persample (with a comparable blank value subtracted). The amount ofradioactivity in the various samples overtime was used to calculatehalf-life in various compartments. Longer chains of heparosan andheparosan conjugates persist in blood after i.v. injection for longperiods of time. Heparosan and heparosan conjugates percolate slowly outof the abdomen after i.p. injection, and thereafter appear in the blood.Heparosan and heparosan conjugates percolate slowly out of the muscleafter i.m. injection and thereafter appear in the blood.

Materials and Methods

Animals: Experiments were performed on male Sprague-Dawley rats (270-345g at time of experiment) purchased from Charles River (Wilmington,Mass.) with a polyurethane catheter implanted into the right jugularvein. Rats were housed under controlled conditions (25° C. 12 hlight/dark cycle) with free access to food and water. Upon arrival, eachrat was placed into a cage and acclimated to the animal facility for atleast 7 days. The institutional Animal Care and Use Committee ofOklahoma University Health Sciences Center approved the animal use forthis protocol (#08-082R).

Test Compounds: The two test compounds (FIG. 2A—100 kDa or FIG. 2B—60kDa polymer) were constructed in a radiolabeled form with I-125 (70Ci/mmol) as decribed previously. The activity of the radiolabel was setat 0.94 μCi per 0.2 ml. The use of I-125 for this study was reviewed andapproved by the OUHSC Radiation Safety Office.

Dosing: Rats were anesthetized by isoflurane inhalation (5-2% to effect)before being administered 0.2 ml of the radiolabeled test compound byi.v. infusion into the right jugular vein. Following compound infusion,the i.v. catheter was flushed with 0.2 ml of sterile saline and then thecatheter was locked with 0.2 ml of sterile heparinized saline (1% v/v,10 U/ml). Rats were then placed into holding cages until beingreanesthetized just before a terminal blood draw and organ collection.

The following groups of rats were dosed:

FIG. 2A - 100 kDa Group 1: 0.5 hr post-dosing n = 2 rats Group 2: 4 hrpost-dosing n = 2 rats Group 3: 8 hr post-dosing n = 2 rats Group 4: 16hr post-dosing n = 2 rats Group 5: 24 hr post-dosing n = 2 rats Group 6:48 hr post-dosing n = 2 rats Group 7: 72 hr post-dosing n = 2 rats

FIG. 2B - 60 kDa Group 1: 0.5 hr post-dosing n = 2 rats Group 2: 4 hrpost-dosing n = 2 rats Group 3: 8 hr post-dosing n = 2 rats Group 4: 16hr post-dosing n = 2 rats Group 5: 24 hr post-dosing n = 2 rats Group 6:30 hr post-dosing n = 2 rats Group 7: 48 hr post-dosing n = 2 rats

Perfusion Groups Group 1: Probe 1, 24 hr post-dosing n = 3 rats Group 2:Probe 2, 16 hr post-dosing n = 3 rats

Sampling and Counting: At the post-dosing time-points listed above ratswere euthanized via a terminal blood draw while under isofluraneanesthesia. Blood was drawn using a 10 cc syringe connected to the i.v.catheter. Median blood volume withdrawn was 10 ml (3 ml, min-11.5 ml,max). For the perfusion groups, 1.5-3.8 ml of blood was withdrawn beforeperfusion. Blood was then transferred into 15 ml Falcon tubes andcentrifuged (3000 rpm for 15 min. at 4° C., Beckman tabletop centrifuge)to separate plasma from blood cells. Once separated, 0.1 ml each ofplasma and blood cells were transferred into plastic culture tubes forsubsequent determination of radioactivity. In addition, liver (3 tubes),spleen (1 tube), kidneys (1 tube/kidney), bladder (empty—1 tube), heart(1 tube), lungs (1 tube/lung), brain (perfusion groups only—2 tubes) andany urine (1 tube) or fresh fecal pellets (1 tube) were collected andprepared for radioactive counting. To determine blood plasma half-lifeand relative distribution of the test compound, the samples were placedinto a gamma counter were the total radioactivity was converted intocounts per minute (CPM). Similar studies were done for i.m. and i.p.injection.

Transcardial Perfusion: At the post-dosing time-points listed above,following blood withdrawal under isoflurane anesthesia, rats wereeuthanized via cardiac perfusion with 200 ml of ice-cold sterile saline.For the perfusion, anesthetized rats were placed on a wire mesh cage topover a sink. Their fore limbs were then taped to the cage top so thatthe chest cavity was exposed. Using forceps to stabilize the skin, aninitial cut with scissors was used to expose the musculature of thechest and upper abdomen. The caudal tip of the sternum was then grasped,and the diaphragm was rapidly punctured with the scissors, followed bycutting through the sternum to expose the heart. Additional cuts to theribs were made with hemostats applied to act as retractors to provide anunobstructed view of the heart. The perfusion system consisted of aperistaltic pump (Masterflex, Cole-Parmer, Vernon Hills, Ill.) used at asetting of ‘7’ with tubing connected to a 16 gauge needle that wasinserted into the left ventricle of the heart which was gently held inplace with forceps to deliver ice-cold sterile saline. The right atriumwas then cut to allow the blood and saline to exit the circulatorysystem. The quality of the perfusion was dependent on the amount of timethe heart remained beating following insertion of the perfusion needleand could be monitored by the loss of color from the tail, hind limbsand liver. At the end of the perfusion, the rat had its organs removedand counted as previously listed.

Data Analysis: pK values: The blood plasma half-life (T_(1/2) or K10) ofthe test compound was determined with WinNonLin software (version 5.2.1)using a Gauss-Newton modeling algorithm (#7), as shown below. Additionalderived pK values including the area under the curve, Cmax, and bodyclearance were calculated by the same software package.

Calculation of total activity: Total activity was determined at eachtime point based on the averaged activity for each listed sample basedon the following calculations:

Estimated total blood volume=[rat body weight (g)/100]×6 ml

Plasma ratio=ml of plasma/ml of total blood withdrawn

Platelet ratio=ml of blood cells/ml of total blood withdrawn

Estimated total plasma volume=plasma ratio×est. total blood volume

Estimated total blood cell volume=platelet ratio×est. total blood volume

Total plasma CPM=plasma CPM/100 μl×1000 μl/1 ml×est. total plasma volume

Total blood cell CPM=blood cell CPM/100 μl×1000 μl/1 ml×est. total bloodcell volume

Total organ CPM=liver CPM+spleen CPM+kidney CPM+bladder CPM+heartCPM+lung CPM

Total Activity=total plasma+total blood cells+total organ

0.5 hr Excreted CPM=0.5 hr urine CPM+0.5 hr fecal CPM

Maximum Activity=0.5 hr Total Activity+0.5 hr Excreted CPM

% Recovery=Total Activity/Maximum Activity

Excreted CPM (all other time points)=Maximum Activity−Total Activity

Calculation of % Relative Activity: For each time-point, the averageactivity for the sample (plasma, blood cells or individual organ) wasdivided by the Total Activity for that time-point. Thus, the total ofall the Relative Activity=100% for each time-point, but the TotalActivity always decreased as the probe was excreted.

Calculation of Perfusion Correction Factor: Data following perfusionfrom individual animals was compared to the values obtained fromnon-perfused animals at the same time-point. The average activity foreach organ was expressed as a percentage of the non-perfused activity ofthe same organ—this percentage was then used as the Perfusion CorrectionFactor and was applied to all time points for the same organ todetermine a Corrected % Relative Activity (% relative activity×perfusioncorrection factor=perfusion corrected % relative activity). For bothprobes, following application of the correction factor, the activitythat was removed from the organs was added to the % relative activityfor the plasma. and blood cells to keep the total at 100% pertime-point.

Pharmacokinetics (pK) Results

pK analysis Probe 1: FIG. 2A shows the curve-fit for the elimination ofthe radioactivity from the plasma for Probe 1. From the softwareanalysis of the data, the T_(1/2) (half-life) or the eliminationconstant was 49.8 hours. The area under the curve (AUC) was 93.3(hr)(pmol/ml) with a Cmax of 1.30 pmol/ml and a clearance of 0.014ml/hr.

pK analysis Probe 2: FIG. 2B shows the curve-fit for the elimination ofthe radioactivity from the plasma for Probe 2. From the softwareanalysis of the data, the T_(1/2) for the elimination constant was 15.4hours. The area under the curve was 33.0 (hr)(pmol/ml) with a Cmax of1.48 pmol/ml and a clearance of 0.040 ml/hr. In addition to long plasmahalf-life, the heparosan molecular weight is stable in the mammalianbloodstream as shown in FIG. 4.

FIG. 15 depicts an analysis of the blood plasma half-life and the bloodabsorption half-life of a radioactive heparosan compound followinginjection thereof into rats. Male Sprague-Dawley rats received two 0.1ml injections of the radioactive heparosan compound (i.m.) in both hindlimb calves. This test compound (100 kDa) was radiolabeled with ¹²⁵I (70Ci/mmol). The activity of the radiolabel was set at 4.0 μCi per 0.2 ml,however testing demonstrated that 10% of the probe was retained withinthe syringes, thus the effective dose was 3.6 μCi. At 1, 6, or 24 hrpost-injection, 0.5 ml of whole blood was collected from the tail vein,while at 48, 72, 96 or 120 hr post-injection the rats were euthanizedwith blood and organs collected to determine distribution of the probe.The blood plasma half-life (T_(1/2) or K10) and the blood absorptionhalf-life (K01) of the test compound was determined with WinNonLinsoftware (version 5.2.1) using a Gauss-Newton modeling algorithm.

FIG. 15 shows the total distribution of the measured radioactivity fori.m. dosing. As shown, the majority of the activity remains in theplasma with relatively constant activity in the red cells and organs.The amount of radioactivity in the different organs dissected from therats was also measured. The activity in the liver was less than 3.0% oftotal activity at all time points. Total activity in the spleen wasalways less than 0.3%. This data indicates that i.m. dosing does notaccumulate in these organs. Activity in the kidneys from remained about2% of the total activity. Activity in the bladder was minimal (<0.2%) atall time points. The probe did not accumulate in either the heart or thelungs as activity in both organs was 1% or less throughout the study.Based on the perfusion tests where residual blood was washed out oforgans, this small amount of radioactivity was due to trapped blood (fori.m. and i.p. studies, no perfusion was performed). Finally, while theinjection site muscle already had a low % total activity (<2%) at thefirst measured time point, the activity further decrease to less than 1%by the end of the experiment. At all time-points when samples wereavailable, both the urine and the fecal pellets were radioactive,demonstrating that with i.m. dosing excretion began by 6 hr post-dosingand continued throughout the testing period. This radioactivity is dueto the residual Bolton-Hunter group attached to a small sugar chain(less than 4 units). Finally, at 96 hr, the brain accounted for 0.2% ofthe total activity and the testis had 0.3% of the total activity forthat rat.

FIG. 16 shows the curve-fit for the elimination of the radioactivityfrom the plasma for i.m. dosing. From the software analysis of the data,the T₂ for the absorption constant was 6.8 hours and the T_(1/2) for theelimination constant was 64.8 hours. The area under the curve was 224.3(hr)(pmol/ml) with a Cmax of 1.70 pmol/ml (33.6% of total dose) at 21.9hr and a clearance of 0.022 ml/hr.

In addition to intravenous and intramuscular injection, radioactiveheparosan was administered to animals via an abdominal injection. MaleSprague-Dawley rats received a single 0.2 ml injection of the 100 kDaheparosan (i.p.) into the peritoneum. At 1, 6, or 24 hr post-injection,0.5 ml of whole blood was collected from the tail vein, while at 48, 72,96 or 120 hr post-injection the rats were euthanized with blood andorgans collected to determine distribution of the probe in a similar tothe i.m. study.

Similar to the i.m. dosing study, the T_(1/2) was at least 2 days whenheparosan was injected i.p. The activity in the liver was less than 3%all time-points. Similar to the i.m. dosing, total activity in thespleen was always less than 0.3%. The activity in the kidneys was 2% orless at all time-points. Activity in the bladder was minimal (<0.1%).The probe did not accumulate in either the heart or the lungs. This dataindicates that i.p. dosing does not accumulate in these organs. At alltime-points with measurements, both the urine and the fecal pellets wereradioactive, demonstrating that i.p. dosing excretion began within thefirst hour post-dosing and continued throughout the testing period.

If the heparosan is not degraded in the extracellular compartments ofthe mammalian body and thus possesses stability, then the chain lengthor molecular weight should remain unchanged. The molecular weight of theheparosan samples from the of i.m. Study (M lanes) that generated theplot of FIG. 15 as well as the samples from i.p. (P lanes) study wasexamined by gel electrophoresis, as shown in FIG. 17. Basically, equalamounts of radioactivity in the plasma from various time points wereconcentrated into a small volume by ultrafiltration (Micron 10 kDa.cut-off). The samples were loaded on an 1X TAE agarose gel (as in FIG.4), electrophoresed, dried and exposed to X-ray film. As a control, theoriginal starting radioactive heparosan (100 kDa) was also loaded (laneS). The data show that the heparosan migrates from the injection sitethrough the various tissues and compartments and gets into blood streamin an intact form with the same molecular weight as the startingpolymer. Even after 5 days (120 hours), the heparosan vehicle remainsintact thus indicating its suitability as a useful vehicle for enhancingtherapeutics.

Analyses of Metabolites after Leaving Bloodstream

Urine: Urine from various time points was extracted with chloroform toremove proteins and concentrated by ultrafiltration as above; the vastmajority of the radioactivity passed through the 3 kDa membranes thusthe heparosan fragments that passed through the kidneys was smaller than3 kDa. This material was then subjected to thin layer chromatography(TLC) on silica plates developed with butanol:acetic acid:water solvent.The plate was then exposed to X-ray film; only small metabolizedfragments (4 sugar units and less or n=2 or less) of the originalheparosan were observed as shown in FIG. 18.

Feces: After injection into rats, heparosan metabolites were excreted infeces over time as shown in FIG. 19. Water extracts of feces fromvarious time points after injection were subjected to ultrafiltrationwith various molecular weight cut-off membranes (3, 10, or 50 kDa;Amicon Microcon) and gamma counting of the retained and elutedfractions. The size of the radioactive metabolites was inferred by theability to penetrate the pores of the membrane; the vast majority of theradioactivity passed through the 3 kDa membranes; thus, the heparosanfragments that passed through the intestines was smaller than 3 kDa.

Reductive Amination Drug-Conjugate Synthesis

The aldehyde of heparosan polymers (e.g, either the natural reducing endor via periodate oxidation) is coupled directly to therapeutic proteinsvia its N-terminal amino groups using reductive animation with Nacyanoborohydride. For adding a single heparosan polymer per polypeptidechain, the reaction is done in 0.1 M Na acetate, pH 5, at 4° to 7° C.;the buffer pH used de-selects the lysine groups with higher pKa (need anunprotonated amine for nucleophilic attack) in favor of the aminoterminus. Lower temperatures (e.g., 4° to 10° C.) is used to preserveprotein folding. Alternatively, reactions at pH 7-9 (e.g., in phosphatebuffer) are used to add multple heparosan chains to the lysines as wellas the N-terminus of the protein cargo. This methodology istherapeutically and commercially successful for proteins likeinterferons and GCSE (Neulasta). A wide variety of chemistries areuseful for successful conjugate synthesis; the basic requirements are(a) there is an appropriate reactive or activated group on the heparosanpolymer vehicle (either short acceptor or longer polymers) that willreact or interact with a group on the cargo (i.e., therapeutic agent ordrug), or if desired, a secondary vehicle (e,g., liposome ornanoparticle), and (b) suitable mild reaction conditions that preservethe integrity and functionality of both the vehicle and the cargo.

Thus, in accordance with the presently disclosed and claimedinvention(s), there has been provided a methodology for HEPylationwherein a heparosan molecule servers as the vehicle for carrying a cargoin a heparosan-conjugate. Although the presently disclosed and/orclaimed inventive concept(s) has been described in conjunction with thespecific drawings and language set forth above, it is evident that manyalternatives, modifications and variations will be apparent to thoseskilled in the art. Accordingly, it is intended to embrace all suchalternatives, modifications and variations that fall within the spiritand broad scope of the invention.

What is claimed is:
 1. A non-immunogenic pharmaceutical drug deliverycomposition for treating a disease condition in a mammalian patient, thepharmaceutical composition comprising: one or more heparosan-drugconjugates, wherein each conjugate comprises: at least one heparosanpolymer, wherein the at least one heparosan polymer is unsulfated; andat least one therapeutic non-peptide drug conjugated to the at least oneheparosan polymer, wherein the at least one therapeutic non-peptide drugtreats a mammalian disease condition and remains active when conjugatedto the at least one heparosan polymer; and a pharmaceutically acceptablecarrier; and wherein the pharmaceutical drug delivery composition is asterile pharmaceutical formulation in a unit dosage format.
 2. Thepharmaceutical drug delivery composition of claim 1, wherein the atleast one therapeutic non-peptide drug does not increaseimmunoreactivity of the at least one heparosan polymer.
 3. Thepharmaceutical drug delivery composition of claim 1, wherein the atleast one therapeutic non-peptide drug is not an adjuvant.
 4. Thepharmaceutical drug delivery composition of claim 1, wherein eachheparosan-drug conjugate comprises a single heparosan polymer conjugatedto the at least one therapeutic non-peptide drug.
 5. The pharmaceuticaldrug delivery composition of claim 1, wherein each heparosan-drugconjugate comprises a plurality of heparosan polymers conjugated to theat least one therapeutic non-peptide drug.
 6. The pharmaceutical drugdelivery composition of claim 1, wherein the at least one heparosanpolymer has a mass in a range of from about 600 Da to about 800 kDa. 7.The pharmaceutical drug delivery composition of claim 1, wherein the atleast one heparosan polymer is unepimerized.
 8. The pharmaceutical drugdelivery composition of claim 1, further defined as comprising aplurality of heparosan-drug conjugates, and wherein the plurality ofheparosan polymers present in the conjugates is polydisperse in size. 9.The pharmaceutical drug delivery composition of claim 1, further definedas comprising a plurality of heparosan-drug conjugates, and wherein theplurality of heparosan polymers present in the conjugates issubstantially monodisperse in size.
 10. The pharmaceutical drug deliverycomposition of claim 1, wherein the at least one heparosan polymer has alinear polymer chain.
 11. The pharmaceutical drug delivery compositionof claim 1, wherein the at least one heparosan polymer has a branchedgeometry.
 12. The pharmaceutical drug delivery composition of claim 1,wherein the at least one heparosan polymer has a half-life of at least15 hours within extracellular compartments of the mammalian patient. 13.The pharmaceutical drug delivery composition of claim 1, wherein atleast one of: (a) the pharmaceutical drug delivery composition exhibitsincreased retention in blood and/or lymphatic circulation of a mammalianpatient when compared to therapeutic non-peptide drug alone; (b) thepharmaceutical drug delivery composition exhibits reduced occurrence ofaccumulation in healthy organs and/or tissues of a mammalian patientwhen compared to therapeutic non-peptide drug alone; (c) thepharmaceutical drug delivery composition exhibits higher accumulation intumors and/or diseased tissues of a mammalian patient when compared totherapeutic non-peptide drug alone; and (d) the at least one heparosanpolymer is further characterized as being substantially non-targeting.14. The pharmaceutical drug delivery composition of claim 1, wherein theat least one heparosan polymer comprises an activated group on the atleast one heparosan polymer to effect the conjugation of the at leastone heparosan polymer to the therapeutic non-peptide drug, and whereinthe reactive group is selected from the group consisting of an aldehyde,alkyne, ketone, maleimide, thiol, azide, amino, carbonyl, sulfhydryl,hydrazide, phosphate, sulfate, nitrate, carbonate, ester, squarate,chelator, and combinations thereof.
 15. The pharmaceutical drug deliverycomposition of claim 1, wherein the at least one heparosan polymer andthe at least one therapeutic non-peptide drug are covalently conjugated.16. The pharmaceutical drug delivery composition of claim 1, wherein theat least one therapeutic non-peptide drug is selected from the groupconsisting of a chemotherapy agent, an antineoplastic agent, a steroid,an antibiotic, an anti-inflammatory agent, an agent that has an actionon a central nervous system of the mammalian patient, an antihistaminic,an antiallergic agent, an antipyretic, a respiratory agent, anantimicrobial agent, an antihypertensive agent, a calcium antagonist, anantipsychotic, an agent for Parkinson's disease, a vitamin, an antitumoragent, a cholinergic agonist, a mydriatic, an antidepressant agent, anantidiabetic drug, an anorectic agent, an antimalarial agent, a hormone,an antiulcerative agent, an anticancer agent, a vaccine antigen, apolynucleotide, a nutrient, a small molecule, and combinations thereof.17. The pharmaceutical drug delivery composition of claim 1, wherein theat least one therapeutic non-peptide drug comprises an anti-canceragent.
 18. The pharmaceutical drug delivery composition of claim 17,wherein the anti- cancer agent comprises cis-platin, actinomycin D,doxorubicin, vincristine, vinblastine, etoposide, amsacrine,mitoxantrone, tenipaside, taxol, colchicine, cyclosporin A,phenothiazines or thioxantheres.